Methods to crosslink biomolecules such as proteins, oligonucleotides and carbohydrates to each other, to radioactive and non-radioactive metal chelates, to drugs and to surfaces have allowed development of both in vitro and in vivo diagnostic assays as well as in vivo therapies. A wide variety of methods have been developed and reviewed (Greg T. Hermanson, Bioconjugate Techniques, Academic Press).
There are a limited number of crosslinking couples, i.e., maleimide/thiol and bromoacetamide/thiol, that are routinely used to prepare conjugates for diagnostic and therapeutic uses. These reagents have limitations in that at high protein concentrations (i.e., >5 mg/mL) protein/protein crosslinking may occur. Also, the maleimido-modified moieties have a limited half-life due to hydrolysis at neutral and basic pH. Incorporation of thiol moieties on biomolecules requires both a coupling and a subsequent activation step. The resultant thiol-modified proteins can readily oxidize to form disulfide polymerized proteins. Also macromolecules containing disulfide bonds, i.e., antibodies, are readily cleaved following activation of the thiol moiety by a reductant. Also, quantitation of the maleimido moiety is somewhat difficult and there is no means to quantify directly the level of conjugation. Therefore, it is advantageous to have a crosslinking couple that does not have these limitations.
Consequently there is a need for crosslinking couples that: bind more efficiently to surfaces; may be controlled to achieve desired crosslinking; do not lead to homobifunctional crosslinking following modification of aggregated proteins; are stable to biological conditions of varying pH and temperature; are stable in solution or when lyophilized; are one step modifications unlike those reagents currently used in the art, e.g., SATA, SPDP type reagents; can be indirectly quantified by a spectrophotometric assay; and can be used to quantify the level of conjugation by spectrophotometric means utilizing the bond formed following conjugation.
Therefore, it is an object herein to provide reagents and methods for crosslinking biomolecules to other biomolecules, polymers, metals or drugs that meet the above needs and have improved properties over known crosslinking reagents and methods.